雷公藤内酯醇抑制脂多糖诱导的PC-12细胞COX-2的表达

 目的　了解雷公藤内酯醇(TP)对PC-12细胞增殖和表达环氧化酶-2(COX-2)及合成前列腺素E₂ (PGE₂ )的影响 ,探讨TP应用于痴呆治疗的可能机制。方法　采用神经生长因子 (NGF)诱导培养PC-12细胞 ,用不同浓度的TP(0,0.28×10^-6,2.8×10^-6,2.8×10^-6mol·L^-1)预处理后 ,加或不加脂多糖(LPS),分别用氚-胸腺嘧啶核苷(³H-TdR)掺入法、竞争酶联免疫吸附试验(ELISA)、半定量逆转录-聚合酶联反应(RT-PCR)法、细胞酶联免疫法及蛋白免疫印迹 (Western-blot)法检测PC-12细胞的增殖活性、细胞培养上清液中PGE² 的水平、细胞COX-2mRNA及蛋白表达水平。同时提取各组细胞蛋白 ,测定其核转录因子 (NF-κB)活性。结果　TP对LPS诱导的PC-12细胞的COX-2mRNA ,蛋白表达 [空白对照与不同浓度下分别为[(0.34±0.12 )×10^-6,(1.92±0.26)×10^-6,(1.78±0.32)×10^-6,(1.14±0.22)×10^-6,(0.48±0.14 )×10^-6mol·L^-1,P <0.01]及PGE₂ 的合成 [分别为(2.28±0.32)×10^-6,(32.86±6.23)×10^-6,(31.28±5.44)×10^-6,(18.43±3.92)×10^-6,(4.18±1.79)×10^-6mol·L^-1,P<0.01]具有不同程度的抑制作用,与TP浓度(0～28×10^-6mol·L^-1)呈明显正相关性(分别为r=0.94和r=0.92),且对LPS激活的PC-12细胞的核转录因子NF-κB活性有明显的抑制作用[分别为(0.16±0.08)×10^-6,(1.39±0.18)×10^-6,(1.09±0.14)×10^-6,(0.52±0.15)×10^-6,(0.28±0.09)×10^-6mol·L^-1,P<1.01]。实验未观察到TP对PC-12细胞增殖的影响。结论TP可显著抑制LPS诱导的PC-12细胞COX-2细胞COX-2的表达及其产物PGE₂的合成,这种作用可能通过TP抑制PC-12细胞核转录因子NF-κB活性而实现。

Inhibition of lipopolysaccharide-induced expression of cyclooxygenase-2 in PC-12 cells by triptolide

OBJECTIVE To explore the effects of triptolide (TP) on cell proliferation,the intracellular expressions of cyclooxygenase-2 (COX-2) and the productions of induced PC-12 cells, which would hopefully add to the understanding of the mechanism of TP in the treatment of prostaglandin E₂(PGE₂) in Alzheimer's disease. METHODS PC-12 cells used in the experiments were cultured with nerve growing factors (NGF),pretreated with TP at various concentrations (0,0.28×10^-6,2.8×10^-6,28×10^-6mol·L^-1),and then treated with or without stimulation of lipopolysaccharide (LPS). The proliferation activities of PC-12 cells were determined by^{3 H-thymine deoxyriboside (3H-TdR) incorporation, and the product of PGE₂ in culture supernatants of PC-12 cells was detected with competitive ELISA and enzyme reduction of nitrate. The expression of COX-2 mRNA in PC-12 cells was analyzed by semi-quantitative RT-PCR, and the expression of COX-2 protein was estimated by Western blot method and cellular enzyme immunoassay. Nuclear factor-B (NF-κB) activity in whole cell extract of PC-12 cells was also measured by an ELISA based method.RESULTS The data showed that TP down regulated the expression of COX-2 mRNA and protein [(0.34±0.12)×10-6,(1.92±0.26)×10-6,(1.78±0.32)×10-6,(1.14±0.22)×10-6,(0.48±0.14)×10-6mol·L-1,P <0.01],and reduced the product of induced PGE₂[(2.28±0.32)×10-6,(32.86±6.23)×10-6,(31.28±5.44)×10-6,(18.43±3.92)×10-6,(4.18±1.79)×10-6mol·L-1,P<0.01] in the LPS-stimulated PC-12 cells This inhibition was positively correlated with TP concentrations(r=0.94and r=0.92, respectively).In addition, NF-κB activity in LPS-stimulated cells was suppressed significantly by TP treatment [(0.16±0.08)×10-6,(1.39±0.18)×10-6,(1.09±0.14)×10-6,(0.52±0.15)×10-6,(0.28±0.09)×10-6mol·L-1,P<0.01]. No effect on the proliferation of PC-12 cells due to TP treatment was observed. CONCLUSION TP mediates the down regulation of expression of COX-2 and product of induced PGE₂ in PC-12 cells, possibly by suppressing the activity of NF-κB.}